Programmed death‐ligand 1 expression in patients with primary or secondary myelofibrosis

Abstract Background It has been described in mice models that myeloproliferative neoplasm (MPN) with JAK2‐V617F mutation has an increased expression of programmed death‐ligand 1 (PD‐L1) in megakaryocytes leading to cancer immune evasion by inhibiting the T‐lymphocytes. Aims To quantify and compare the PD‐L1 expression on bone marrow (BM) of patients with MPN JAK2 positive, negative, and normal controls. Methods We collected BM of patients with MPN JAK2 positive, negative and normal controls from 1990 to 2019. We also created a scoring system to quantify PD‐L1 expression in megakaryocytes. Results We obtained 14 BM with JAK2 positive PMF, 5 JAK2 negative PMF, and 10 patients with normal BM biopsies. PD‐L1 expression was higher in the JAK2 positive group compared with the control group with a score of 212.6 versus 121.1 (t‐value 2.05, p‐value 0.025). In addition, the score was higher in the PMF group regardless of JAK2 mutational status when compared with the control group with score of 205.9 versus 121.1 (t‐value 2.12, p‐value 0.021). There was no difference in the PD‐L1 score between the JAK2 negative versus the control group 187.2 versus 121.1 (t‐value 1.02, p‐value 0.162). Conclusion These findings suggest that PMF patients with a JAK2 mutation have a higher PD‐L1 expression in megakaryocytes compared with the control group. We postulate that the combination of checkpoint and JAK2 inhibitors may be an active treatment option in JAK2 mutated PMF given the higher PD‐L1 expression.


| INTRODUCTION
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by constitutional activation of the JAK-STAT signaling pathway and bone marrow (BM) fibrosis, which leads to decreased peripheral blood counts, proinflammatory state, and a potential for transformation to acute myeloid leukemia.The median age at presentation is 65 years. 1,2JAK2-V617F is the most common mutation in PMF and is found in 50%-60% of patients. 3The only curative treatment option currently available is allogeneic stem cell transplant.
However, most patients are ineligible because of advanced age and comorbidities. 4Ruxolitinib and fedratinib, JAK2 inhibitors, are the only FDA approved treatment for intermediate/high risk PMF patients. 5,6However, those drugs have their limitations and only improve symptoms and decrease splenomegaly, without an overall survival benefit. 5,6Therefore, there is a significant unmet need for treatment options in this patient population.
Prestipino et al. discovered that mice models with JAK2-V617F mutated MPN generally have an increased expression of programmed death-ligand 1 (PD-L1) that leads to cancer immune evasion by inhibiting the antitumor effect of the T lymphocytes against cancer cells. 7eckpoint inhibitors are monoclonal antibodies that block the interaction between PD-L1 and its receptor, allowing the immune system to fight cancer cells with an enhanced antitumor response.Pembrolizumab was the first checkpoint inhibitor approved by the FDA for patients with metastatic nonsmall-cell lung cancer with >50% PD-L1 expression in tumor cells by immunohistochemistry stain.In this subset of patients, pembrolizumab was more effective than systemic chemotherapy. 8 this paper, we compared the PD-L1 expression among patients with JAK2-mutated PMF versus JAK2-unmutated PMF patients versus normal controls without no PMF or JAK2 mutation.

| Study population
We collected BM biopsies of patients with PMF done at Tulane Medical Center from 1990 to 2019.All these patients had a known JAK2 status and a well-preserved specimen for adequate PD-L1 staining.We only used the initial BM biopsy obtained to diagnose patients with PMF.We collected 10 additional samples of patients that came to clinic with transient cytopenias from benign hematologic conditions.These patients had normal karyotype, FISH panel for myelodysplastic syndrome, and a negative 64-gene next generation sequencing (NGS) myeloid mutation panel.An institutional review board approval was obtained before collecting these BM samples and all participants gave informed consent.The authors analyzed the data to which all authors had access.All relevant participant data was deidentified and shared as appropriate in the text.

| PD-L1 immunohistochemistry staining
We used the FDA approved test PD-L1 IHC 28-8 pharmDx to assess the PD-L1 expression on the BM biopsies.This is a qualitative immunohistochemical assay, which uses monoclonal mouse anti-PD-L1 Clone 22C3 intended for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded tissues of different cancers such as nonsmall cell lung cancer and gastric/gastroesophageal junction adenocarcinoma.This test is used to identify patients who may be treated with pembrolizumab.
BM sections of 4-5 μm were made with tissues mounted on microscope slides then placed in a 58 ± 2 C oven for 1 h.The slides were stained with the PD-L1 IHC 22C3 pharmDx reagent, and the samples were incubated.Finally, all the slides were numbered and labeled with codes.

| Interpretation of PD-L1 expression
The slides were reviewed and scored independently by two clinical pathologists specialized in interpreting PD-L1 expression in solid tumors.PD-L1 was scored according to the quantity and intensity in the megakaryocytic lineage of cells.The quantity of PD-L1 expression was graded from 0 to 100% while the intensity was graded from 1+ to 3+, according to the level of intensity.The result was a PD-L1 score calculated by multiplying percentage by intensity of PD-L1 in the BM megakaryocytes.This score will be detailed further in the statistical analysis section.

| RESULTS
We studied the BM biopsies of 29 patients in total; 14 patients had JAK2-mutated PMF, 5 JAK2-unmutated PMF, and 10 controls with negative BM biopsies.The median age of the whole group was 57 years, with 34% males and 66% females.The main clinical characteristics of the myelofibrosis patients are described in the Table 1.
PD-L1 expression and intensity are described in Table 2.We have a few examples how the PD-L1 expression and intensity was quantified in the BM biopsies, as shown in Figures 1-4.
The average PD-L1 expression score for the JAK2-mutated PMF group was 212.57, the JAK2-unmutated PMF group 187.2, and the control group 121.1.There was a statistically significant difference between the PD-L1 score between the JAK2-mutated PMF group versus the control group (t-value 2.05 and p-value of .025)and when we compared the PMF group regardless of the JAK2 status versus the control group (t-value 2.12 and p-value of .021).However, there was not a statistically significant difference when we compared the PD-L1 expression between the JAK2 negative versus the control group (t-value 1.02 and p-value .162).
Myelofibrosis patients with a PD-L1 score <250 had a median overall survival of 130 months versus PD-L1 score >250 of 64 months (hazard ratio 2.63, 95% CI; 0.82-8.4;p = .09),it was numerically longer but not statistically significant as shown in Figure 5.

| DISCUSSION
The presented results show that PD-L1 score was higher in the PMF group versus the control group regardless of JAK2 mutation status, which turned out to be statistically significant.This helps build on the data reported by Prestipino et al. that showed oncogenic JAK2 activity led to STAT phosphorylation, which in turn enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2 mutant cells. 7In addition, PD-L1 expression was higher on primary cells isolated from patients with JAK2-mutated MPNs as compared with healthy individuals and declines upon JAK2 inhibition. 7Moreover, Lee et al. were able to demonstrate that PD-L1 expression was significantly associated with overt myelofibrosis and JAK2 mutational status. 9Moreover, in the previously mentioned study, there were four patients who were found to have a particularly high PD-L1 expression that also harbored the JAK2 mutation. 9 F I G U R E 2 It represents a bone marrow BX of a 28-year-old woman with unexplained anemia in the slide the pathologist counted 29 megakaryocytes that looked normal and they did not highlight the programmed death-ligand 1 (PD-L1) stain, for that reason, the expression and intensity were score as 0. PD-L1 score was 0.
F I G U R E 1 It represents the bone marrow BX of a 76-year-old woman with newly diagnose JAK2-mutated PMF, and the megakaryocytes are hyperplastic with all of them expressing programmed death-ligand 1 (PD-L1) with the highest intensity.In this slide, the pathologist counted 97 cells.Expression was quantified as 97% (39 + 39 + 19), and intensity was very bright and for that reason, it was given the highest score of 3+.The PD-L1 score was 291.
F I G U R E 3 It represents a bone marrow BX of a 73-year-old woman with JAK2-unmutated programmed death-ligand 1 (PD-L1) that reveals some hyperplastic megakaryocytes.In this slide, the pathologist counted 54 cells.Expression was quantified as 78% (55.6 + 18.5 + 3.7), and intensity was bright but not as intense as in the Figure 1, for that it was scored as 2+.The PD-L1 score was 156.
F I G U R E 4 It represents a bone marrow BX of a 60-year-old man with JAK2-unmutated primary myelofibrosis with hyperplastic megakaryocytes.In this slide, the pathologist counted 31 cells.Expression was quantified as 100% (38.7 + 48.4 + 12.9), and intensity was very bright, for that, it was scored as 3+.The programmed death-ligand 1 score was 300.
of expression and intensity of PD-L1 on the 29 bone marrow biopsies.Main characteristics of patients with myelofibrosis.